JPH0693834B2 - Thermostable nicotinamide adenine dinucleotide kinase and method for producing the same - Google Patents

Description

The present invention relates to a thermostable nicotinamide adenine dinucleotide kinase and a method for producing the same by a microorganism.

[Conventional technology]

Nicotinamide adenine dinucleotide kinase (hereinafter referred to as NAD kinase) is a transferase with EC number 2.7.2.23, and nicotinamide adenine dinucleotide (hereinafter referred to as NAD) and adenosine triphosphate (hereinafter referred to as NAD kinase) ATP
Abbreviated) and nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP) and adenosine
It catalyzes the formation of diphosphoric acid (hereinafter abbreviated as ADP). NAD kinase is a useful enzyme for the production of NADP from NAD. NADP
Is known to be an important substance as a coenzyme for dehydrogenase, and is very useful as a clinical test reagent.

NAD kinase is used in animal livers such as pigeons, rats, and rabbits and yeast, Azotobacter vinelandi (Azotobacter
It is known to exist in the microbial cells such as vinelandii).
However, conventionally, NAD kinase is a relatively unstable enzyme, and is easily deactivated, especially in a cell-free state, and it has been considered impossible to mass-produce the enzyme.

[Problems to be solved by the invention]

Therefore, if a heat-stable NAD kinase can be obtained, it will be very effective for the large-scale purification of the enzyme and the production of NADP from NAD.

An object of the present invention is to provide a novel thermostable NAD kinase and a method for producing the same.

[Means for solving problems]

Briefly describing the present invention, the present invention relates to a thermostable NAD kinase and a method for producing the same, and the first invention of the present invention relates to a thermostable NAD kinase having the following physicochemical properties.

(1) Action It acts on nicotinamide adenine dinucleotide and adenosine triphosphate to generate nicotinamide adenine dinucleotide phosphate and adenosine diphosphate by a transphosphorylation reaction.

(2) Substrate specificity It works best on adenosine triphosphate. It does not act on adenosine diphosphate and adenosine monophosphate.

(3) Optimum pH and pH stability The optimum pH is 7.2 and the stable pH range is 4.0 to 8.5.

(4) Optimal reaction temperature and thermal stability The optimum reaction temperature is 50 ° C, and it is stable up to 65 ° C after 15 minutes of treatment.

(5) Molecular weight is about 200,000 (by gel filtration method).

(6) Inhibitor It is inhibited by SH reagents such as 5,5'-dithiobis-2-nitrobenzoic acid, p-chloromercuribenzoic acid, N-ethylmaleimide and iodoacetic acid.

A second invention of the present invention relates to a method for producing a thermostable NAD kinase of the first invention, which relates to Corynebacterium
bacterium), which is a thermostable nicotinamide adenine dinucleotide kinase-producing bacterium belonging to the genus
It is characterized in that thermostable nicotinamide adenine dinucleotide kinase is collected from the culture.

As a result of detailed examination of NAD kinases of various strains, the present inventors have found that Corynebacterium strains produce thermostable NAD kinases. The present invention is based on this new finding.

The thermostable NAD kinase obtained by the present invention is
Since it is stable even at 0 to 65 ° C treatment, it is possible to denature and remove the contaminating enzyme activity by heat treatment.
As detailed in No. 199, the phosphatase activity that interferes with the reaction in the production of NADP from NAD can be simply removed. Furthermore, by removing the contaminating proteins, nucleic acids and the like by denaturing and coagulating them by heat treatment, the purification operation of NAD kinase can be greatly simplified. Thus, the present invention provides an industrially useful NAD kinase and an industrial production method thereof.

Next, the present invention will be described in more detail.

Any strain may be used as long as it belongs to the genus Corynebacterium and has the ability to produce a thermostable NAD kinase. A particularly suitable strain is Corynebacterium flatscumfaciens (Coryne
bacterium flaccumfaciens) AHU-1622 (FERM BP-73
1) is mentioned.

The strain is the Japanese Federation
Of Culture Collections Of Microorganisms Catalog Of Cultures 1979
〔The Japanese Federation of Culture Collections o
f Microorganisms (JFCC) Catalog of Cultures 1979
THIRD EDITION], Japan Scientific
Society Press (Japan Scientific Societies
Press) is the same strain as Corynebacterium flatscumfaciens AHR 1622 described in 1980, and the strain is a strain obtained from Hokkaido University Faculty of Agriculture.

As a medium for these microorganisms, sucrose, glucose, saccharides such as soluble starch, and the like as carbon sources, organic acids such as citric acid and malic acid, and hydrocarbons can be used, and as a nitrogen source, Meat extract, peptone, polypeptone, glutamic acid, aspartic acid, etc. and organic and inorganic sources such as ammonium sulfate and ammonia are used. Further, potassium phosphate such as dibasic potassium phosphate and dibasic potassium phosphate, magnesium salts such as magnesium sulfate or magnesium chloride, metal ions such as iron, manganese, zinc and calcium, and trace amounts necessary for growth of each strain. A substance added with an appropriate amount is used. The culture is carried out under conditions such as shaking culture or aeration-agitation culture. Culture temperature is 20
-40 ° C, preferably 25-37 ° C, pH 5.5-9.0, preferably 7.0-8.0. Cultivation time is usually 5 to 50 hours. NAD kinase is produced in the cells and the culture broth cultured by the above method.

Purification of NAD kinase can be carried out by salting out with ammonium sulfate or precipitation with an organic solvent such as ethanol, and ion exchange chromatography such as DEAE-Sephadex, SP-Sephadex, DEAE-Toyopearl, SP-Toyopearl.

The unit U of enzyme activity is the enzyme titer that converts 1 nmole of NAD to NADP in 1 minute.

Next, the method for producing NAD kinase of Corynebacterium flatscumfaciens AHU-1622 (FERM BP-731) and the properties of the enzyme will be described with reference to Examples.

Example Corynebacterium fratcumfaciens
AHU-1622 (FERM BP-731 ) of NAD kinase production and properties DL- malic acid _{1g / dl, MgSO 4 · 7H} 2 O 0.03g / dl, citrate 0.
3g / dl, K ₂ HPO ₄ 1.5g / dl, Na (NH ₄ ) HPO ₄ 0.53g / dl, KOH 0.
Add 500 ml of a medium consisting of 6 g / dl, item extract 0.1 g / dl and peptone 1 g / dl (pH 7.0) to a 2 liter Erlenmeyer flask and store at 120 ° C for 15
Sterilize for minutes. The above medium was inoculated with Corynebacterium fratcumfaciens AHU-1622, and 200 at 200C for 48 hours.
Incubate with shaking at rpm. The culture-finished solution was centrifuged to obtain bacterial cells, which were washed and then suspended in 100 ml of 100 mM phosphate buffer containing 10 mM 2-mercaptoethanol, and the cells were suspended at 5 ° C.
The cells are crushed with an ultrasonic crusher at 9 KHz (Kubota Manufacturing Co., Ltd.) for 10 minutes below. The supernatant obtained by centrifuging the disrupted solution was used as a crude enzyme solution.

The enzymatic properties of the NAD kinase thus obtained were examined below.

(1) Substrate specificity It acts best on adenosine triphosphate. It does not act on adenosine diphosphate and adenosine monophosphate.

(2) Molecular weight is about 200,000 (by gel filtration method).

(3) Inhibitor It is inhibited by SH reagents such as 5,5'-dithiobis-2-nitrobenzoic acid, p-chloromercuribenzoic acid, N-ethylmaleimide and iodoacetic acid.

(4) pH stability of NAD kinase Dilute the enzyme solution with buffers adjusted to various pH values of 100 mM, and
After treatment at 0 ° C. for 5 hours, the remaining activity was measured. pH
2.4-6.9 is acetate buffer, pH 7.8-8.8 is Tris buffer, pH
Borate buffer was used for 9.5 to 11.0. The result is first
As shown in the figure. From FIG. 1, it can be seen that the stable pH is 4.0 to 8.5. In FIG. 1, ∘, Δ, □, and × marks indicate that acetic acid buffer, phosphate buffer, Tris buffer, and borate buffer are used, respectively.

(5) Optimal pH of NAD kinase reaction The reaction was carried out at 30 ° C and various pH values using the same enzyme solution as in (4). The buffer used is the same as that used in the pH stability experiment of (4). The results are shown in Fig. 2. Second
The figure shows that the optimum pH is 7.2. In FIG. 2, ∘, Δ, □, and × marks indicate that acetic acid buffer, phosphate buffer, Tris buffer, and borate buffer are used, respectively.

(6) Optimum reaction temperature of NAD kinase The reaction temperature is 20, 30, 35, 40, using the same enzyme solution as in (4).
The enzyme activity was measured at 45, 50, 55, 60, 65, 70 ℃. The results are shown in Fig. 3. From FIG. 3, it can be seen that the optimum reaction temperature is 50 ° C.

(7) Heat resistance of NAD kinase The same enzyme solution as in (4) was previously prepared for 20,25,30,35,40,45,50,55,
The residual activity was measured after treatment between 15 powders at 60, 65 and 70 ° C and cooling. The results are shown in Fig. 4. From FIG. 4, it can be seen that NAD kinase is stable up to 65 ° C.

For comparison, the Brevibacterium ammoniagenes (Brev
The thermostability of NAD kinase obtained from the bacterium (ibacterium ammoniagenes) was similarly examined. From Fig. 4, it can be seen that the heat resistance of Brevibacterium NAD kinase is up to 40 ° C. As described above, it was confirmed that the NAD kinase of Corynebacterium fratcumfaciens AHU-1622 is extremely thermostable.

In FIG. 4, ∘ and Δ indicate the NAD kinase originating from Corynebacterium fratcumfaciens according to the present invention and Brevibacterium ammonia dienes as a comparative example, respectively.

[Examples] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

Example 1 Corynebacterium fratcumfaciens AHU-1622 (FERM BP-731) is used as an inoculum. DL- malic acid _{1g / dl, MgSO 4 · 7H} 2 O0.03g / dl, citric acid 0.3g / dl, K ₂ HP
Medium 500 consisting of O ₄ 1.5 g / dl, Na (NH ₄ ) HPO ₄ 0.53 g / dl, KOH 0.6 g / dl, yeast extract 0.1 g / dl, peptone 1 g / dl (pH 7.0)
Add 2 ml Erlenmeyer flask and sterilize at 120 ° C for 15 minutes. One platinum loop of the above strain was inoculated into the above medium, and cultured at 30 ° C. for 48 hours with shaking.

After culturing, all of the culture solution is inoculated into 15 liters of the main culture medium in a 30 liter jar fermenter. The composition of the main culture medium is KM-70 (Shin-Etsu Chemical Co., Ltd.)
Manufactured by K.K.) was used.

The main culture was carried out at 30 ° C., 350 rpm, aeration amount 1 / medium 1.1 minutes for 24 hours. NAD kinase activity is 0 per 1 ml of culture medium.
It was 39U.

To collect NAD kinase from the culture-completed liquid, the liquid was centrifuged to obtain bacterial cells, which were washed with cold water and then
100 mM phosphate buffer containing mM2-metacaptoethanol 1.
After suspending in 5 liters, the cells were crushed with an ultrasonic crusher at 9 KHz [Kubota Manufacturing Co., Ltd.] at 5 ° C. or lower for 10 minutes to obtain a crushed cell solution. Ammonium sulfate was added to the supernatant obtained by centrifuging this crushed solution to make ammonium sulfate 80% saturated,
The precipitate was collected by centrifugation. This precipitate was added to 10 mM
After dissolving in 100 mM phosphate buffer containing mercaptoethanol, it was dialyzed against copper buffer overnight.

Then, each 20 ml of the obtained enzyme solution was equilibrated with 100 mM phosphate buffer containing 10 mM 2-mercaptoethanol, Toyopearl HW-55 (manufactured by Toyo Soda Co., Ltd.) column (diameter 4c
The gel was run at m, length 95 cm). Fractions eluted with NAD kinase activity were collected, concentrated with Collodion bag and lyophilized. NAD kinase was harvested with an activity yield of approximately 40%. The activity of the obtained NAD kinase preparation was 0.12 U / mg, and the activity per absorbance at 280 nm was 10 U.

Example 2 As the carbon source of the medium, sucrose was used instead of DL-malic acid, and other conditions were the same as in Example 1 for culturing. The NAD kinase activity of the obtained culture broth is 1
It was 1.63 U per ml.

The enzyme was purified from the culture solution by salting out with ammonium sulfate and gel filtration in the same manner as in Example 1, and then freeze-dried to obtain an enzyme powder. The activity of the obtained NAD kinase preparation was 0.51 U / mg, and the activity per absorbance at 280 nm was 12 U.

〔The invention's effect〕

As described above in detail, according to the present invention, a new thermostable NAD kinase-producing ability was found in a strain belonging to the genus Corynebacterium, and an industrial production method of this enzyme could be established.

INDUSTRIAL APPLICABILITY The present invention has an excellent effect in that it has developed a technique for producing a thermostable NAD kinase useful for producing NADP from NAD.

[Brief description of drawings]

FIG. 1 is a graph showing the pH stability of the NAD kinase of Corynebacterium flatscumfaciens AHU-1622 of the present invention. FIG. 2 shows the optimum reaction of NAD kinase of Corynebacterium flatscumfaciens AHU-1622 of the present invention.
It is a graph which shows pH. FIG. 3 is a graph showing the optimum reaction temperature of NAD kinase of Corynebacterium flatscumfaciens AHU-1622 of the present invention. Figure 4 shows the NAD of Corynebacterium fratcumfaciens AHU-1622.
1 is a graph showing the thermostability of NAD kinase of Kinase and Brevibacterium ammoniagenes.

Claims (2)

[Claims] 1. A thermostable nicotinamide adenine dinucleotide kinase having the following physicochemical properties. (1) Action It acts on nicotinamide adenine dinucleotide and adenosine triphosphate to produce nicotinamide adenine dinucleotide phosphate and adenosine diphosphate by a transphosphorylation reaction. (2) Substrate specificity It works best on adenosine triphosphate. It does not act on adenosine diphosphate and adenosine monophosphate. (3) Optimum pH and pH stability The optimum pH is 7.2 and the stable pH range is 4.0 to 8.5. (4) Optimal reaction temperature and thermal stability The optimum reaction temperature is 50 ° C, and it is stable up to 65 ° C after 15 minutes of treatment. (5) Molecular weight of about 200,000 (by gel filtration method). (6) Inhibitor It is inhibited by SH reagents such as 5,5'-dithiobis-2-nitrobenzoic acid, p-chloromercuribenzoic acid, N-ethylmaleimide and iodoacetic acid. 2. Corynebacterium
Characterized in that a heat-resistant nicotinamide adenine dinucleotide kinase-producing bacterium belonging to the genus is cultivated in a medium, and a heat-resistant nicotinamide adenine dinucleotide kinase having the following physicochemical properties is collected from the culture. A method for producing a thermostable nicotinamide adenine dinucleotide kinase. (1) Action It acts on nicotinamide adenine dinucleotide and adenosine triphosphate to produce nicotinamide adenine dinucleotide phosphate and adenosine diphosphate by a transphosphorylation reaction. (2) Substrate specificity It works best on adenosine triphosphate. It does not act on adenosine diphosphate and adenosine monophosphate. (3) Optimum pH and pH stability The optimum pH is 7.2 and the stable pH range is 4.0 to 8.5. (4) Optimal reaction temperature and thermal stability The optimum reaction temperature is 50 ° C, and it is stable up to 65 ° C after 15 minutes of treatment. (5) Molecular weight of about 200,000 (by gel filtration method). (6) Inhibitor It is inhibited by SH reagents such as 5,5'-dithiobis-2-nitrobenzoic acid, p-chloromercuribenzoic acid, N-ethylmaleimide and iodoacetic acid.